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TC Basics
Please try to sign up for hoods/centrifuges with accurate time estimates! TC ettiquette Sign up for hood time on the sign-up sheets located on each hood. The top of the block=the start of that block of time. IE: If I sign up during the 8:00 block, then my time starts at 8:00 and ends at 8:30. Before using the hood: *Remember to always wear gloves, and wipe down the hood before using it! *Spray anything that goes in the hood with EtOH before putting in the hood! (except for plates of cells) *If EtOH is low or empty, it's easy to make more! Just fill the bottle approx 3/4 to the top with 100% EtOH (by the chemical weighing area, in the large canister with a pump). Fill to the top with water, and invert to mix a few times. *Check the liquid waste--if it's full, take it to the sink, add bleach, and dump it down the drain. Add more bleach to the container and hook it back up. After using the hood: *Wipe down the hood! *Make sure tips, glass pipettes, and serological pipettes are all re-stocked. Make sure gas and vaccuum are off. *Double check the liquid waste--change it if necesary. *Make sure you leave nothing of yours behind. Cells/Media We work mainly with primary keratinocytes (KCs), melanocytes, fibroblasts, and a variety of cell lines including 293Ts, phoenix cells, 3T3s, and cancer cell lines. Media should be stored at 4C whenever it's not in use. Warm it up in the 37C waterbath before using it (warm for about 15minutes). After making a bottle of media, it lasts for about 1 month. After a month it's best to toss the remaining media and make fresh media. Keratinocytes are cultured in 50/50 media. 50/50 is 50% 154 media and 50% KC serum-free media. *1 bottle 154 for KCs + supplement (from behind abx, in foil) *1 bottle KCSFM + suppl *Abx *Pen/Strep Mix in 1L container, put back into the two media containers, label Fibroblasts, 293Ts, phoenix cells, 3T3s are cultured in 10% DMEM: *1 bottle DMEM *1 50mL vial FBS (makes DMEM 10% FBS) *pen/strep *abx Thaw FBS, and mix everything together in the DMEM bottle. Label and store at 4C. Changing media *warm media for 10-15minutes *spray bottle with ethanol, spray hood with ethanol *aspirate media from cell dish using a glass pipette *replace with appropriate media **6cm plate: 3-4mLs **10cm plate: 10-12mL **15cm plate: 25mL Washing KCs of melanocytes *usually done right before splitting cells, but can wash KCs of melanocytes without passaging KCs *warm trypsin, 10% DMEM, 50/50 *aspirate media *add trypsin to KCs and incubate at 37C for ~2-3minutes (1-2mL per 10cm plate) *aspirate trypsin *wash with 10% DMEM **if going to split KCs, then simply aspirate trypsin, and add more trypsin, continue splitting *wash with PBS (optional) *replace 50/50 media Splitting (passaging) Cells *warm trypsin, 10% DMEM, 50/50 (if necesary) *aspirate media *add trypsin to plate (1-2mL for 10cm, 5-7mL for 15cm) *incubate in 37C for 5mins *tap sides if necessary to loosen cells (optional: view dish in microscope to see if most cells are in suspension) *use pipette to collect trypsin-cell suspension, transfer to 50mL conical *wash plate with at least same volume 10% DMEM (used 2mL trypsin, use at least 2mL 10% DMEM) *spin cells down in large centrifuge for 5mins (1200rpm) *aspirate DMEM *resuspend in appropriate media *count if necessary, seed onto new plates!